Int. J. Pharm. Sci. Rev. Res., 29(1), November – December 2014; Article No. 25, Pages: 131-134
ISSN 0976 – 044X
Research Article
Anticancer Activity of Allium sativum (Bulb) Polyphenolic Compound
Rajeev Nema, Sarita Khare, Alka Pradhan
Sarojini Naidu Government Girls Post Graduate (Autonomous) College, Shivaji Nagar, Bhopal, M.P., India.
*Corresponding author’s E-mail: rrsht.nema@gmail.com
Accepted on: 25-08-2014; Finalized on: 31-10-2014.
ABSTRACT
The aim of the present research is to determine the use of Allium Sativum (Bulb) Polyphenolic Compound activity on MCF-7, A549
and PA-1 cancer cell line (breast, lung and ovary cancer respectively). Hydro alcoholic (1:1) sample of Allium Sativum (Bulb) were
prepared and tested for their cytotoxic activities against cancer cell lines (MCF-7, A-549 and PA-1) with standard Doxorubicin. The
most essential reason of this study is to estimate cytotoxicity of certain important Indian medicinal plants with facilitate of MTT
assay. Concentrations are set of each plant extract which are 100 µg/ml, 10 µg/ml , 0.1 µg/ml, 0.01 µg/ml and 5-10×10 3 cells/ml are
taken into each well which are exposed to different Concentrations of Allium Sativum (Bulb) for 96 hr and then treated with MTT.
For MTT absorbance in use at 570 nm. From IC50 values of MTT assay of Allium Sativum (Bulb) for MCF7, A549 and PA1 cancer cell
lines, from this it may conclude that Allium Sativum (Bulb) shows efficient cytotoxicity on MCF-7 (6 ± 1 µg ) than PA-1(15 ± 1 µg) and
A459 (28 ± 1 µg ) cancer cell line.
Keywords: Allium Sativum, Anticancer activity, Soxhlet extraction.
tetrazole)8, Distilled Water, Dimethyl sulphoxide (DMSO)9,
etc.
INTRODUCTION
llium sativum L. (GARLIC)
A
Scientific classification
Kingdom: Plantae; clade: Angiosperms; clade:
Monocots;
Order:
Asparagales;
Family:
Amaryllidaceae; Subfamily: Allioideae; Genus: Allium;
Species: A. sativum L.
Description
Allium sativum is a bulb. It grows up to 0.5 m (2ft) in
height. Its hardiness is USDA Zone. It produces
hermaphrodite flowers. Pollination occurs by insects and
bees.
Biological and Medicinal Property
The effects of garlic on health with its possible preventive
effects on the development of cancer in humans have
been mentioned in previous reviews. In addition A.
sativum has free radical scavenging activity, immune
system modulation and direct cytotoxic effect on cancer
cells.1 Now a day’s numerous epidemiological, clinical and
laboratory studies have demonstrated the role of garlic in
cancer prevention2-3 especially in relation to digestive
tract cancers, including esophageal and stomach cancers.4
There is also promising research evaluating the use of
garlic in leukemic melanoma5 and neuroblastoma cell
6
lines.
MATERIALS AND METHODS
Requirements
Laminar air flow, Autoclave, N2 liquid container, CO2
incubator, Inverted microscope, Filtration assembly,
Hemocytometer, Centrifuge machine, Micropipette,
Soxhlet, Spectrophotometer.
Plant Material Collection
Allium Sativum (Bulb) of plants ware collected from
Bhopal during month of October. Than dried up under the
shed dry for six week furthermore crush it.
Soxhlet Extraction: Hydroalcoholic (1:1)
Soxhlet extraction is only required where the desired
compound has a limited solubility in a solvent, and the
impurity is insoluble in that solvent. Allium Sativum (Bulb)
were extracted in Soxhlet Apparatus using Hydroalcoholic
solvent (1:1).
Phytochemical Analysis
The hydroalcolic extract of Allium Sativum (Bulb) was
tested for the presence of various phytoconstituents such
as Carbohydrate, Starch, Protein, Aminoacids, Steroids,
Flavonoids, Alkaloids, Tannins, Phenolic Compounds,
oxalic acid and inorganic compounds. All phytochemical
tests were done as per the procedure given in the
standard book (Practical Pharmacognosy by C.K. Kokate).
The FT-IR analysis of the Allium Sativum (Bulb) extract
was done and the functional groups associated were
determined.
Column chromatography
Alcohol 70%, 100% Alcohol, MEM media (Minimal
7
Essential Media) (Eagle H 1959), Trypsin , MTT (3-(4,5Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, a
After phytochemical analysis bioactive compounds
present in extract was separated out by column
chromatography in a proper solvent system. Column
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Int. J. Pharm. Sci. Rev. Res., 29(1), November – December 2014; Article No. 25, Pages: 131-134
chromatography was performed on a classic 20 cm long ×
2 cm diameter glass column packed with 50 g Silica gel of
60-120 mesh size as stationary phase and crude drug
were further subjected to column chromatography [CC]
and eluted with specific solvent to obtain pure
compounds. Silica gel for column chromatography was
used as stationary phase. The flow rate used was 5
ml/min. Three and four elutes for each solvent were
taken.
ISSN 0976 – 044X
colorimeter. Analyze data of test with standard drug and
plot graph.14, 18
RESULTS AND DISCUSSION
Phytochemical Evaluation: The results of preliminary
phytochemical evaluation are summarized in Table 1.
Table 1: The results of preliminary phytochemical
evaluation
Spectrophotometric Determination of Total Flavonoid
Content (TFC)
Natural product
Test performed
Result
Carbohydrate
Molish test
+Ve
Total flavonoid contents were measured by Aluminum
chloride colorimetric assay. Hydroalcoholic extracts that
has been adjusted to come under the linearity range and
different dilution of standard solution of Quercetin (10100µg/ml) were added to 3ml of water. To the above
mixture, 0.1ml of 5% C4H4O6KNa.4H2O (Potassium Sodium
L-(+) - Tartrate Tetrahdrate) was added. After 5 minutes,
0.1ml of 10% AlCl3 was added and the total volume was
made up to 3 ml with distilled water. It was left at room
temperature for 30 min after which the absorbance of the
reaction mixture was measured at 430nm with a single
beam spectrophotometer (Systronic).10-11
Starch
Iodine
-Ve
Protein
Millions
+Ve
Amino acid
Cysteine test
+Ve
Steroid
Salkowski test
+Ve
Isolation of Human Cancer Cells
Human cancer cells are isolated from the patients and
characterized at cellular and molecular levels. Isolated
cells are cultivated in specialized mediums and specialized
incubators to provide them physiological conditions
required for the growth.12-13
Cell Line
The sub culturing of the primary culture gives rice to cell
lines. The term continuous cell line implies the indefinite
development of the cell in the successive sub culturing.
On the other hand, finite cell lines symbolize the death of
cell after several subcultures. The considered cell lines are
MCF-7 14-15 (barest cancer), A-549 16 (lung cancer) and PA1 17(ovary cancer).
Assay Performed
MTT Assay Method
Flavonoids
+Ve
Alkaloid
Mayer’s test
+Ve
Tannic and phenolic compound
%5 Fecl3 test
+Ve
Oxalic acid
+Ve
Inorganic acid
Sulphate test
+Ve
Column chromatography
Column chromatography of Allium Sativum (Bulb) was
performed on a classic 20 cm long × 2 cm diameter glass
column packed with 50 g Silica gel of 60-120 mesh size as
stationary phase and Allium Sativum (Bulb) crude drug
were further subjected to column chromatography [CC]
and eluted with specific solvent chloroform methanol
(1:2) to obtain pure compounds.
FT-IR Spectral Analysis
The FT-IR spectrum of the Allium Sativum (Bulb) leaves
extract recorded the number of peaks lying between
3330.14 cm-1, 2943.57 cm-1, 2831.65 cm-1, 17.64 cm-1,
1453.30cm-1,1453.30 cm-1,1239.30cm-1,1111.50 m1,1021.76 cm- 1,751.81 cm-1, and 662.91 cm-1
respectively. This finding will help in further research in
the investigation of medicinally active chemical
compounds present in Allium Sativum (Bulb).
Quantification
Laminar air flow was prepared. Dilutions of concentration
100 µg/ml, 10 µg/ml, 1 µg/ml, 0.1 µg/ml, 0.01 µg/ml from
stock solution (test drug +DMSO) having concentration
10mg/ml is done. Then normal count on haemocytometer
before seeding the cells in plate was done. 10µl from
each conc. in 4wells i.e. 20 wells for one drug was added.
Plate contained 5-10×10 3 cells/ml into each well of 96well culture plate. The cells were incubated for 96 hr in
CO2 incubator. After it cells are incubated with basal
medium containing 0.5 mg/ml MTT in CO2 incubator at
37°C for appropriate duration of time. The medium is
aspirated, and the formazan product is solubilized with
dimethyl sulfoxide (DMSO). Absorbance at 570 nm is
measured for each well using a microplate reader on
Current study revealed the flavonoid contents of Allium
Sativum (Bulb). (Quercetin standard plot: y =0.0966x,
19
R2=0.9878). On the basis that calibration curve was
plotted by preparing the Quercetin solutions at
concentrations 12.5 mg/ml-1. Total flavonoid content of
the extracts was expressed as percentage of flavonoid in
plant extract 4.35 µ.g./ml equivalents per dry weight of
sample and take notice of y=0.002x+0.0148, R2 =0.993.
Cell Line
MCF-7
A-549
PA1
Sample Code
AS
AS
AS
IC50 (mg/ml)
6±1
20±1
11±1
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Int. J. Pharm. Sci. Rev. Res., 29(1), November – December 2014; Article No. 25, Pages: 131-134
MTT Assay Result
ISSN 0976 – 044X
DISCUSSION
Figure 1: MCF7 Cells were treated with hydro alcoholics
extract of Allium Sativum dissolved in DMSO at 0.01, 0.1,
1, 10, 100 conc. Cells were subjected to MTT within 1 hr24 hr. Response of MCF7 Cell to Allium Sativum For %
Inhibition on Y-axis and Concentration on X-axis.
Allium Sativum (Bulb) extracts was investigated. The
invitro cytotoxic potentiality was investigated as the
ability of Allium Sativum (Bulb) extracts to inhibit tumour
cell line growth. With this investigation we had also
focused on angiogenesis. The studied cell lines are MCF7,
A549 and PA1. After exposure of cells to Allium Sativum
(Bulb) extract cell line were treated with MTT Dye which
results into the live cells convert the MTT to purpled
colour formazan crystals, which are soluble in Dimethyl
sulphoxide (DMSO). After solubilization of crystals then
absorption is taken on spectrophotometer at 570 nm.
With respect to readings the graphs were plotted for %
inhibition on Y-axis and Conc. of drug on X-axis. The
readings were directly converted into percentage. from
this it may conclude that Allium Sativum (Bulb) shows
efficient cytotoxicity on MCF-7 (6 ± 1 µg ) than PA-1(15 ±
1 µg) and A459 (28 ± 1 µg ) cancer cell line where
Standard drug was used for IC50 of Doxorubicin MCF-7
500nm, A549- 550nm, PA-1- 580nm.
CONCLUSION
Figure 2: A549 Cells were treated with hydro alcoholics
extract of Allium Sativum dissolved in DMSO at 0.01, 0.1,
1, 10, 100 conc. Cells were subjected to MTT within 1 hr24 hr. Response of A549 Cell Allium Sativum For %
Inhibition on Y-axis and Concentration on X-axis.
In the present time herbal products are considered to be
symbols of protection in comparison to the synthetic
product that are regarded as unsafe to human life and
environment. Although herbs had been priced for their
medicinal importance. But now everyday phytochemical
and pharmacological studies are conducted on different
parts of plants. More research can be done to investigate
the unknown and unexplored potential of these plants.
Further analysis of these plants (active compounds) can
be carried out by way of making use of different analytical
methods such as HPTLC, HPLC, NMR and UV
spectrophotometer analysis.
Acknowledgement: The authors express gratitude Amit
Deshpande Deshpande Lab., and Pvt., Ltd., Bhopal, MP.
India.
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Figure 3: PA1 Cells were treated with hydro alcoholics
extract of Allium Sativum dissolved in DMSO at 0.01, 0.1,
1, 10, 100 conc. Cells were subjected to MTT within 1 hr24 hr. Response of PA1 Cell to Allium Sativum For %
Inhibition on Y-axis and Concentration on X-axis.
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Source of Support: Nil, Conflict of Interest: None.
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